The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that is mutated in patients with cystic fibrosis (CF), disruptin fluid and ion balance in multiple epithelial tissues. In the lung, failure of mucociliary clearance facilitates the establishment of drug-resistant bacterial biofilms, despite advanced antibiotic and pulmonary clearance strategies. As a result, chronic lung infection and inflammation are currently the major causes of CF morbidity and mortality, limiting lifespan to <40 years. ~90% of CF patients carry one or two copies of the ?F508 allele, which encodes a protein that is inefficiently folded, shows limited channel activity, and is rapidly degraded. Compounds have been identified that address the folding and channel defects. Neither provides significant benefit as a monotherapy, but in combination they produce significant improvement in lung function (?FEV1 >10%) in 25% of ?F508 homozygous patients. To reach more patients and increase the functional response, we propose the early-stage pharmacological validation of a novel translational strategy to address the remaining defect - the degradation of rescued ?F508-CFTR. No clinical trials include compounds specifically designed to increase CFTR stability at the apical membrane. Having identified the CFTR-Associated Ligand (CAL) as a key mediator of CFTR degradation, we have localized a critical binding interface, designed peptides that block it, and shown that they act as first-in-class 'stabilizers' of functional ?F508-CFTR in polarized CF bronchial epithelial cells. Preclinical advancement of our inhibitor-of- CAL (iCAL) approach is currently limited by lead affinity, delivery, and limited data on the extent of additioal rescue compared to combination therapies currently in clinical trials. Here, we propose to leverage preliminary advances in all three areas. Our new data confirm substantial additivity for a cell-permeable iCAL in concert with VX-809. With a validated target, multiple lead chemistries, a strong suite of functional assays, and structural and biochemical expertise, we propose an integrated structure-activity approach to optimize CAL inhibitors and confirm their therapeutic potential. In Aim 1, using existing peptide inhibitors with state-of-the-art ex vivo CF patient intestinal current measurement and in vitro mucociliary transport assays, we will establish a functional pipeline to evaluate combinations of CAL inhibitors with potentiator and corrector molecules approved or in late-stage clinical trials. In Aim 2, we propose to validate and optimize the bioactivity of iCAL peptides for systemic or inhaled therapies. In Aim 3, building on a suite o biochemical assays, we describe structure-based strategies to improve the affinity and selectivity of small-molecule inhibitors. Together these studies will determine threshold parameters governing CFTR rescue. Having joined forces to produce proof of concept for CFTR stabilizers, our interdisciplinary and tightly coordinated collaboration is well positioned to obtan second-generation CAL inhibitors with demonstrated efficacy and biological tolerability, while developing a compelling portfolio for the further pharmacological development of this novel therapeutic target.